1、BSI Standards PublicationPD CEN ISO/TS 18867:2015Microbiology of the foodchain Polymerase chainreaction (PCR) for the detectionof food-borne pathogens Detection of pathogenicYersinia enterocolitica andYersinia pseudotuberculosis(ISO/TS 18867:2015)PD CEN ISO/TS 18867:2015 PUBLISHED DOCUMENTNational f
2、orewordThis Published Document is the UK implementation of CEN ISO/TS18867:2015.The UK participation in its preparation was entrusted to TechnicalCommittee AW/9, Microbiology.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not pu
3、rport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2016. Published by BSI StandardsLimited 2016ISBN 978 0 580 83730 2ICS 07.100.30Compliance with a British Standard cannot confer immunity fromlegal obligatio
4、ns.This Published Document was published under the authority of theStandards Policy and Strategy Committee on 31 March 2016.Amendments issued since publicationDate Text affectedPD CEN ISO/TS 18867:2015TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN ISO/TS 18867 October 20
5、15 ICS English Version Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection of food-borne pathogens - Detection of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis (ISO/TS 18867:2015) Microbiologie de la chane alimentaire - Raction de polymrisation en
6、chane (PCR) pour la dtection de micro-organismes pathognes dans les aliments - Dtection des Yersinia enterocolitica et Yersinia pseudotuberculosis pathognes (ISO/TS 18867:2015)Mikrobiologie der Lebensmittelkette - Polymerase-Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorganismen in Lebensm
7、itteln - Nachweis von pathogenen Yersinia enterocolitica und Yersinia pseudotuberculosis (ISO/TS 18867:2015) This Technical Specification (CEN/TS) was approved by CEN on 29 May 2015 for provisional application. The period of validity of this CEN/TS is limited initially to three years. After two year
8、s the members of CEN will be requested to submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard. CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available promptly at nati
9、onal level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cr
10、oatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
11、United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No.
12、 CEN ISO/TS 18867:2015 EPD CEN ISO/TS 18867:2015CEN ISO/TS 18867:2015 (E) 2 Contents Page European foreword . 3 PD CEN ISO/TS 18867:2015CEN ISO/TS 18867:2015 (E) 3 European foreword This document (CEN ISO/TS 18867:2015) has been prepared by Technical Committee ISO/TC 34 “Food products“ in collaborat
13、ion with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the secretariat of which is held by DIN. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying
14、 any or all such patent rights. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugosl
15、av Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO/TS 18867:2015 h
16、as been approved by CEN as CEN ISO/TS 18867:2015 without any modification. PD CEN ISO/TS 18867:2015ISO/TS 18867:2015(E)Foreword vIntroduction vi1 Scope . 12 Normative references 13 Terms and definitions . 14 Principles . 14.1 General . 14.2 Microbial enrichment 24.3 Nucleic acid extraction . 24.4 Am
17、plification and detection 24.5 Isolation 25 Reagents 25.1 General . 25.2 Culture media . 25.2.1 General 25.2.2 Diluent 25.2.3 Enrichment media 25.2.4 Selective solid medium . 45.2.5 Potassium hydroxide in saline solution, KOH . 55.3 Nucleic acid extraction . 55.4 Reagents for PCR . 55.5 Primers and
18、probes. 56 Apparatus and equipment 56.1 General . 56.2 Equipment for sample preparation prior to enrichment . 66.3 Equipment for microbial enrichment 66.4 Equipment for nucleic acid extraction 66.5 Equipment for real-time PCR 67 Sampling 68 Procedure. 68.1 Sample preparation prior to enrichment . 68
19、.1.1 General 68.1.2 Preparation of the sample . 68.2 Microbial enrichment 78.2.1 Pathogenic Y. enterocolitica . 78.2.2 Y. pseudotuberculosis 78.2.3 Pathogenic Y. enterocolitica and Y. pseudotuberculosis 78.3 Isolation of colonies, optional 78.3.1 Pathogenic Y. enterocolitica . 78.3.2 Y. pseudotuberc
20、ulosis 78.3.3 Process controls . 88.4 Nucleic acid extraction . 88.5 PCR amplification . 88.5.1 General 88.5.2 PCR controls . 88.6 Confirmation of the PCR product 88.6.1 General 88.6.2 Interpretation of the PCR result . 89 Test report . 9Annex A (normative) PCR detection and isolation of pathogenic
21、Y. enterocolitica (see ISO 2015 All rights reserved iiiContents PagePD CEN ISO/TS 18867:2015ISO/TS 18867:2015(E)Figure A.1) 10Annex B (informative) Real-time PCR for detection of Y. enterocolitica 11Annex C (informative) Detection and isolation of Y. pseudotuberculosis .21Annex D (informative) Simul
22、taneous detection of pathogenic Y. enterocolitica and Y. pseudotuberculosis using multiplex real-time PCR 26Bibliography .29iv ISO 2015 All rights reservedPD CEN ISO/TS 18867:2015ISO/TS 18867:2015(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of nationa
23、l standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. Internation
24、al organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended f
25、or its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.o
26、rg/directives).Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be
27、 in the Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions
28、related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is the European Committee for Standardization (CEN) Tech
29、nical Committee CEN/TC 275, Food analysis Horizontal methods, in collaboration with Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement). ISO 2015 All rights reserved vPD CEN IS
30、O/TS 18867:2015ISO/TS 18867:2015(E)IntroductionYersinia enterocolitica and Yersinia pseudotuberculosis are zoonotic bacterial pathogens causing food-borne infection (yersiniosis) in humans worldwide. The main reservoir for pathogenic Y. enterocolitica is domestic pigs3and for Y. pseudotuberculosis a
31、 wide range of domestic and wild animals such as rodents, deer, birds, and various farm animals serve as potential reservoirs.4Some of the biotypes of Y. enterocolitica are associated with human infection. In contrast, all Y. pseudotuberculosis are considered potentially pathogenic to humans.9The ch
32、romosomally located gene ail (attachment invasion locus) is present in all bio(sero)types of Y. enterocolitica associated with disease and a variant of it is also present in Y. pseudotuberculosis.8The ail gene is the target gene used for detection in this Technical Specification, and the developed p
33、rimer/probe sets target different sites of the ail gene for the two pathogens.781314vi ISO 2015 All rights reservedPD CEN ISO/TS 18867:2015Microbiology of the food chain Polymerase chain reaction (PCR) for the detection of food-borne pathogens Detection of pathogenic Yersinia enterocolitica and Yers
34、inia pseudotuberculosis1 ScopeThis Technical Specification specifies two horizontal methods for detection of the pathogenic bioserotypes of Y. enterocolitica and one for detection of Y. pseudotuberculosis by using real-time PCR-based methods. The described methods allow for the detection of the two
35、pathogens in enrichments and allow the isolation of colonies. Y. pestis, the causative agent of bubonic and pneumonic plague harbours a variant of the ail gene as well and will be detected by the same primer/probe set as Y. pseudotuberculosis. However, Y. pestis is normally not associated with food.
36、 This Technical Specification is applicable to products for human consumption, animal feeding stuffs, and environmental samples.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated referen
37、ces, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examinati
38、on Part 1: General rules for the preparation of the initial suspension and decimal dilutionsISO 10273, Microbiology of food and animal feeding stuffs Horizontal method for the detection of presumptive pathogenic Yersinia enterocoliticaISO 20837, Microbiology of food and animal feeding stuffs Polymer
39、ase chain reaction (PCR) for the detection of food-borne pathogens Requirements for sample preparation for qualitative detectionISO 20838, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens Requirements for amplification and detec
40、tion for qualitative methodsISO 22119, Microbiology of food and animal feeding stuffs Real-time polymerase chain reaction (PCR) for the detection of food-borne pathogens General requirements and definitionsISO 22174, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for
41、the detection of food-borne pathogens General requirements and definitions3 Terms and definitionsFor the purpose of this document, the following terms and definitions given in ISO 22174 and ISO 22119 apply.4 Principles4.1 GeneralThe method comprises the following consecutive steps:a) Microbial enric
42、hment (4.2);TECHNICAL SPECIFICATION ISO/TS 18867:2015(E) ISO 2015 All rights reserved 1PD CEN ISO/TS 18867:2015ISO/TS 18867:2015(E)b) Nucleic acid extraction (4.3);c) Amplification and detection (4.4);d) Isolation (4.5).4.2 Microbial enrichmentThe number of pathogenic Y. enterocolitica and Y. pseudo
43、tuberculosis bacterial cells is increased by growth in a non-selective or semi-selective liquid nutrient medium.4.3 Nucleic acid extractionBacteria cells are separated from the nutrient broth, lysed, and the nucleic acid extracted for use in the PCR reaction.4.4 Amplification and detectionThe extrac
44、ted nucleic acid is amplified using a probe-based real-time PCR. Detection of the target sequence is achieved by monitoring a clear increase in the fluorescence signal above the cycle threshold, Ct.NOTE Probe-based real-time PCR combines amplification, detection, and confirmation of the target DNA.4
45、.5 IsolationAfter a PCR-positive result is obtained, the target organism can be isolated by using culture methods as described in this Technical Specification.5 Reagents5.1 GeneralFor the stages in 4.1 b)-c), molecular grade reagents and consumables suitable for molecular biology shall be used as gi
46、ven in ISO 20837 and ISO 20838.Requirements are specified in ISO 20838.The following media and reagents should be used.5.2 Culture media5.2.1 GeneralSee ISO 7218 and ISO 11133 for the preparation, production, and performance testing of culture media.5.2.2 DiluentSee ISO 6887-1 and the relevant part
47、of ISO 6887 dealing with the product to be examined.5.2.3 Enrichment media5.2.3.1 Tryptone-soya broth supplemented with yeast, TSBY5.2.3.1.1 CompositionPancreatic digest of casein 17,0 g2 ISO 2015 All rights reservedPD CEN ISO/TS 18867:2015ISO/TS 18867:2015(E)Papaic digest of soyabean meal 3,0 gSodi
48、um chloride, (NaCl) 5,0 gDibasic potassium phosphate, (K2HPO4) 2,0 gGlucose 2,5 gYeast extract 6,0 gWater 1 000 ml5.2.3.1.2 PreparationDissolve the above ingredients in 1 000 ml distilled water. Adjust the pH, if necessary, so that after sterilization it is pH 7,3 0,2. Dispense the medium into tubes
49、 or flasks of suitable capacity to obtain portions appropriate for the test samples. Sterilize for 15 min at 121 C 1 C.Store the medium in the dark at room temperature and not longer than 4 weeks.Alternatively, use dehydrated Tryptone soya broth (TSB) 30 g/l supplemented with 0,6 % yeast extract, pH 7,3 0,2.5.2.3.2 Peptone-sorbitol-bile-salt broth, PSB155.2.3.2.1 CompositionPeptone 5,0 gSorbitol 10,0 gSodium chloride, (NaCl) 5,0 gDisodium hydrogen phosphate (Na2HPO4) 8,23 gSodium dihydrogen phosph
